Polypeptide with appetite regulating activity

ABSTRACT

The present invention relates to a polypeptide with appetite regulating function/activity, a nucleic acid construct encoding the polypeptide and a method of producing the polypeptide. The invention further relates to recombinant vectors comprising the nucleic acid construct encoding the polypeptide, recombinant host cells comprising the nucleic acid construct or the recombinant vector.

FIELD OF INVENTION

[0001] The present invention relates to a polypeptide with appetiteregulating function/activity, a nucleic acid construct encoding thepolypeptide and a method of producing the polypeptide.

[0002] The invention further relates to recombinant vectors comprisingthe nucleic acid construct encoding the polypeptide, recombinant hostcells comprising the nucleic acid construct or the recombinant vector, atransgenic animal or plant containing and expressing the nucleic acidconstruct, an appetite regulating composition comprising thepolypeptide, and the use of the polypeptide for regulating appetite.

[0003] The polypeptide has appetite regulating activity/function inmammals, including humans.

BACKGROUND OF THE INVENTION

[0004] It has been known that certain tumors when implanted into ratsafter a period of growth suddenly induce severe anorexia and adipsia(lack of eating and drinking) in the animal, whereas closely relatedtumor lines do not, Madsen et al., Scand. J. Clin. Invest. Supplement220: 27-36.

[0005] The aim of this invention has been to find the factor(s)responsible for this characteristic phenotype.

[0006] Cocaine and Amphetamine Regulated Transcript (CART) was detectedas one of several compounds that was selectively expressed in anorecticversus non-anorectic secondary cultures of glucagonomas. In situhybridisation analysis of CART mRNA expression has shown a decreasedlevel of CART mRNA in the nucleus arcuatus and nucleus paraventricularisof the rat hypothalamus following fasting. Similarly, CART mRNA in thearcuate nucleus of Zucker rats (fa/fa) was strongly decreased whencompared to heterozygote controls (fa/+) as measured by in situhybridisation. Thus, CART mRNA in the arcuate nucleus demonstrates apattern of change inverse to that known for NPY. The latter findingprovides a strong linkage between the expression of CART and biologicalfactors involved in food intake.

[0007] A polypeptide of at least 30 amino acids was found by Spiess etal., 1981, Biochemistry 20:1982-1988 as an HPLC peak when purifyingsomatostatine from sheep hypothalamus. The isolated polypeptide was theC-terminal (IPI-CART) portion of CART. However, no biological functionwas associated with this molecule.

[0008] The mature CART peptide has so far not been isolated andcharacterised. A transcript to be upregulated in rat brain aftertreatment with cocaine and amphetamine relating to CART was cloned. Thiscloning indicates that the peptide may exist in a long form consistingof 102 amino acid residues or in a short form consisting of 89 aminoacid residues (Douglass, J. et al. J. Neurosci. 15, 2471-2481, 1995).

[0009] The same group found the human gene and cDNA for CART. Only theshort form exists in humans (Douglass and Daoud (1996), Gene 169:241-245).

[0010] In 1995 Amgen disclosed methods of reducing or preventing neurondegeneration and promoting regeneration and restoration of functioninduced by CART (WO 96/34619).

SUMMARY OF THE INVENTION

[0011] The aim of this invention has been to find the factor(s)responsible for the above described characteristic phenotype.

[0012] It has now been found that a polypeptide with the sequence SEQ IDNo. 1 and fragments thereof have appetite regulating activity/function:Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Pro-Arg-Arg-Gln-Leu-Arg-Ala-Pro-Gly-Ala-Val-Leu-Gln-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Als-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-ArgGly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu

[0013] Furthermore, it has been found that the following polypeptideshave appetite regulating activity/function: SEQ ID No. 2:Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQID No. 3:Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Als-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQID No. 4:Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 5:Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 6:Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 7:Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 8:Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Als-Gly-Glu-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No. 9:Cys-Asp-Als-Gly-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQID No.10:Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu SEQ ID No.11:Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu

[0014] The peptides SEQ ID Nos. 5 to 11 are considered to be novel perse and are constituting a part of the invention.

[0015] In a preferred embodiment of the present invention the cysteineresidues of the above peptides SEQ ID Nos. 1 to 11 are linked bydisulphide bonds in the configuration I-III, II-V and IV-VI when thecysteines are numbered from the N-terminal. These peptides are alsoconsidered to be novel per se and are constituting a part of theinvention.

[0016] In the present context, the term “appetite regulatingactivity/function” is intended to mean any activity/function whichsuppresses appetite e.g. by inducing a feeling of satiety or byinhibiting the sensation of hunger. The appetite regulatingactivity/function may be measured according to the test methodsdescribed in Example 9 or 20

[0017] In another aspect, the invention relates to nucleic acidconstructs comprising a nucleotide sequence encoding a CART polypeptideor a fragment or variant thereof with appetite regulatingactivity/function.

[0018] In a further aspect, the invention relates to nucleic acidconstructs encoding a polypeptide with a sequence selected from thesequences SEQ ID Nos. 1 to 9 such as the sequences SEQ ID Nos. 1 to 9 inwhich the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe N-terminal end.

[0019] In a further aspect, the invention relates to recombinant vectorscomprising the nucleic acid constructs and recombinant host cellscomprising the nucleic acid constructs or the vectors.

[0020] In a further aspect, the invention relates to a method ofproducing a CART polypeptide or a fragment or a variant thereof withappetite regulating activity/function which method comprises cultivatinga host cell as defined above in a suitable culture medium underconditions permitting expression of the nucleic acid construct andrecovering the resulting polypeptide from the culture medium/cell.

[0021] In a further aspect, the invention relates to transgenic animalsor transgenic plants comprising the nucleic acid construct as definedabove as well as methods of producing a CART polypeptide or a fragmentor a variant thereof with appetite regulating activity/function usingsuch transgenic animals or transgenic plants.

[0022] In still a further aspect, the invention relates to an antibodycapable of specifically binding to a CART polypeptide or a fragment or avariant thereof with appetite regulating activity/function, such as apolypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to9, e.g. the sequences SEQ ID Nos. 1 to 9 in which the cysteine residuesare linked by disulphide bonds in the configuration I-III, II-V andIV-VI when the cysteines are numbered from the N-terminal end. In apreferred embodiment of the invention the antibody is monoclonal and theinvention furthermore relates to hybridomas producing such monoclonalantibodies.

[0023] In a further aspect, the invention relates to appetite regulatingcompositions comprising the polypeptides as defined above and apharmaceutically acceptable carrier and the use of the polypeptides forthe preparation of medicaments for the regulation of appetite. In apreferred embodiment of the invention the medicaments are used for thetreatment of obesity.

[0024] Furthermore, the invention relates to a method for the regulationof appetite comprising administering to a subject in need thereof aneffective amount of a polypeptide as defined above. In a further aspect,the invention relates to the use of CART or CART fragments or variantsto identify a functional receptor and the subsequent use of the CARTreceptor to identify receptor agonists with appetite regulatingactivity.

[0025] In a further aspect, the invention relates to compounds thatupregulate the CART expression and thereby regulate appetite.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1: Cloning of CART.

[0027]FIG. 2. Expression in E. coli. Fusion toGlutathione-S-transferase.

[0028]FIG. 3: Expression in E. coli. Fusion to Thioredoxin.

[0029]FIG. 4: Expression of CART in yeast.

[0030]FIG. 5: Sequence of heterologous protein expression cassette ofplasmid pEA182 and pEA183 in yeast.

[0031]FIG. 6: Preparation of FXa-digest of the thioredoxin-CART fusionprotein.

[0032]FIG. 7: Analytical HPLC of CART yeast supernatant H-372.

[0033]FIG. 8: SP-Sepharose Column.

[0034]FIG. 9: Analytical HPLC of CART fragment, pool A.

[0035]FIG. 10: Analytical HPLC of CART fragment, pool B.

[0036]FIG. 11: Analytical HPLC of CART fragment, pool C.

[0037]FIG. 12: Primary and secondary structure of “IPI-CART” showing theI-III, II-V and IV-VI disulphide bond configuration.

[0038]FIG. 13: Effect of fasting on CART mRNA expression.

[0039]FIG. 14: CART mRNA in Zucker rat arcuate nucleus and heterozygotecontrols.

[0040]FIG. 15: S. cerevisiae plasmid for the expression and secretion ofGlu-Glu-Ile-Asp-CART(55-102). TPI-prom. and TPI-term. are S. cerevisiaetriosephosphate isomerase transcription promoter and terminatorsequences, respectively. TPI S. pombe is the Schizosaccharmyces pombetriosephosphate isomerase gene. Only restriction sites relevant for theplasmid construction have been indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0041] This invention is based on the unexpected and surprisingdiscovery that CART polypeptide has been found to possess appetiteregulating function/activity. In the present context the term“polypeptide” is understood to include a mature protein or a precursorform thereof as well as a functional fragment thereof which essentiallyhas the activity of the full-length polypeptide.

[0042] Furthermore, the term “polypeptide” is intended to includehomologues of said polypeptide. Such homologues comprise an amino acidsequence exhibiting a degree of identity of at least 60%, preferably 80%with the amino acid sequences shown in SEQ ID Nos. 1-9. The degree ofidentity may be determined by conventional methods, see for instance,Altshul et al., Bull. Math. Bio. 48: 603-616, 1986, and Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, twoamino acid sequences are aligned to optimize the alignment scores usinga gap opening penalty of 10, a gap extension penalty of 1, and the“blosum 62” scoring matrix of Henikoff and Henikoff, supra.

[0043] Alternatively, the homologue of the polypeptide may be oneencoded by a nucleotide sequence hybridizing with an oligonucleotideprobe prepared on the basis of the polypeptide sequences shown in SEQ IDNos. 1-9 .

[0044] In a further aspect the invention relates to a variant of thepolypeptide of the invention. The variant is one in which one or moreamino acid residues in one or more positions have been substituted byother amino acid residues.

[0045] Homologues of the present polypeptide may have one or more aminoacid substitutions, deletions or additions. These changes are preferablyof a minor nature, that is conservative amino acid substitutions that donot significantly affect the folding or activity of the protein, smalldeletions, typically of one to about 30 amino acids, small amino- orcarboyxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or asmall extension that facilitates purification, such as a poly-histidinetract, an antigenic epitope or a binding domain. See in general Ford etal., Protein Expression and Purification 2: 95-107, 1991. Examples ofconservative substitutions are within the group of basic amino acids(such as arginine, lysine, histidine), acidic amino acids (such asglutamic acid and aspartic acid), polar amino acids (such as glutamineand asparagine), hydrophobic amino acids (such as leucine, isoleucine,valine), aromatic amino acids (such as phenylalanine, tryptophan,tyrosine) and small amino acids (such as glycine, alanine, serine,threonine, methionine).

[0046] It will be apparent to persons skilled in the art that suchsubstitutions can be made outside the regions critical to the functionof the molecule and still result in an active polypeptide. Amino acidsessential to the activity of the polypeptide of the invention, andtherefore preferably not subject to substitution, may be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,Science 244, 1081-1085, 1989). In the latter technique mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for biological activity (e.g. appetite regulation)to identify amino acid residues that are critical to the activity of themolecule. Sites of ligand-receptor interaction can also be determined byanalysis of crystal structure as determined by such techniques asnuclear magnetic resonance, crystallography or photoaffinity labelling.See, for example, de Vos et al., Science 255: 306-312, 1992; Smith etal., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.

[0047] The homologue may be an allelic variant, i.e. an alternative formof a gene that arises through mutation, or an altered polypeptideencoded by the mutated gene, but having substantially the same activityas the polypeptide of the invention. Hence mutations can be silent (nochange in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence.

[0048] The homologue of the present polypeptide may also be a specieshomologue, i.e. a polypeptide with a similar activity derived fromanother mammalian species eg. rat, mouse, sheep or human.

[0049] Furthermore, homologues of said polypeptide may be found in othertisssues such as the brain and pancreas.

[0050] A homologue of the polypeptide may be isolated by preparing agenomic or cDNA library of a cell of the species or tissue in question,and screening for DNA sequences coding for all or part of the homologueby using synthetic oligonucleotide probes in accordance with standardtechniques, e.g. as described by Sambrook et al., Molecular Cloning:ALaboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989, or by means of polymerase chain reaction (PCR) usingspecific primers as described by Sambrook et al. and Saiki at al.,Science 239 (1988) 487-491.

[0051] It may be preferred to provide the polypeptide in a highlypurified form, i.e. greater than 90% pure, more preferably 95% and mostpreferably 99% pure, as determined by analytical HPLC.

[0052] The currently preferred polypeptides of the invention are theones comprising the amino acid sequences shown in SEQ ID Nos. 1-9.

[0053] Nucleic Acid Construct

[0054] As used herein the term “nucleic acid construct” is intended toindicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNAor RNA origin. The term “construct” is intended to indicate a nucleicacid segment which may be single or double stranded, and which may bebased on a complete or partial naturally occurring nucleotide sequenceencoding a polypeptide of interest. The construct may optionally containother nucleic acid segments.

[0055] The nucleic acid construct of the invention encoding thepolypeptide of the invention may suitably be of genomic or cDNA origin,for instance obtained by preparing a genomic or cDNA library andscreening for DNA sequences coding for all or part of the polypeptide byhybridization using synthetic oligonucleotide probes in accordance withstandard techniques (cf. Sambrook et al., supra). For the presentpurpose, the DNA sequence encoding the polypeptide is preferably ofmammalian origin, i.e. derived from a genomic DNA or cDNA library. Morepreferably, the DNA sequence may be of rodent origin, e.g. rat or miceorigin. Even more preferably, the DNA sequence may be of human origin.

[0056] The nucleic acid construct of the invention encoding thepolypeptide may also be prepared synthetically by established standardmethods, e.g. the phosphoamidite method described by Beaucage andCaruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the methoddescribed by Matthes et al., EMBO Journal 3 (1984), 801-805. Accordingto the phosphoamidite method, oligonucleotides are synthesized, e.g. inan automatic DNA synthesizer, purified, annealed, ligated and cloned insuitable vectors.

[0057] Furthermore, the nucleic acid construct may be of mixed syntheticand genomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

[0058] The nucleic acid construct may also be prepared by polymerasechain reaction using specific primers, for instance as described in U.S.Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

[0059] As template for the PCR cloning we used the same double strandedcDNA preparation as described in example 1 (from MSL-A-AN). The PCRreaction, 25 cycles: 60 sec 94° C. 30 sec 52° C. 60 sec 72° C.

[0060] The nucleic acid construct is preferably a DNA construct whichterm will be used exclusively in the following.

Recombinant Vector

[0061] In a further aspect, the present invention relates to arecombinant vector comprising a DNA construct of the invention. Therecombinant vector into which the DNA construct of the invention isinserted may be any vector which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

[0062] The vector is preferably an expression vector in which the DNAsequence encoding the polypeptide of the invention is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid or viral DNA, or maycontain elements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the polypeptide.

[0063] The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.

[0064] Examples of suitable promoters for directing the transcription ofthe DNA encoding the polypeptide of the invention in mammalian cells arethe SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864),the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222(1983), 809-814) or the adenovirus 2 major late promoter.

[0065] An example of a suitable promoter for use in insect cells is thepolyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBSLett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen.Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosisvirus basic protein promoter (EP 397 485), the baculovirus immediateearly gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S.Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

[0066] Examples of suitable promoters for use in yeast host cellsinclude promoters from yeast glycolytic genes (Hitzeman et al., J. Biol.Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, N.Y., 1982), or the TPI1 (U.S. Pat. No. 4,599,311)or ADH2-4c (Russell et al., Nature 304 (1983), 652-654) promoters.

[0067] Examples of suitable promoters for use in filamentous fungus hostcells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J.4 (1985), 2093-2099) or the tpiA promoter. Examples of other usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters.

[0068] Examples of suitable promoters for use in bacterial host cellsinclude the promoter of the Bacillus stearothermophilus maltogenicamylase gene, the Bacillus licheniformis alpha-amylase gene, theBacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilisalkaline protease gen, or the Bacillus subtilis xylosidase gene, or bythe phage Lambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters.

[0069] The DNA sequence encoding the polypeptide of the invention mayalso, if necessary, be operably connected to a suitable terminator, suchas the human growth hormone terminator (Palmiter et al. cit.) or (forfungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnightet al., op. cit.) terminators. The vector may further comprise elementssuch as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elbregion), transcriptional enhancer sequences (e.g. the SV40 enhancer) andtranslational enhancer sequences (e.g. the ones encoding adenovirus VARNAs).

[0070] The recombinant vector of the invention may further comprise aDNA sequence enabling the vector to replicate in the host cell inquestion. An example of such a sequence (when the host cell is amammalian cell) is the SV40 origin of replication.

[0071] When the host cell is a yeast cell, suitable sequences enablingthe vector to replicate are the yeast plasmid 2μ replication genes REP1-3 and origin of replication.

[0072] When the host cell is a bacterial cell, sequences enabling thevector to replicate are e.g. the Col E1 origin of replication as inpUC19 or pBR322 or the p15A origin of replication as in pACYC184 whenthe bacterium is E. coli. When the bacterium is B. subtilis the originof replication from e.g. pUB110 is often used.

[0073] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. Forfilamentous fungi, selectable markers include amdS, pyrG, argB, niaD,sC.

[0074] To direct a polypeptide of the present invention into thesecretory pathway of the host cells, a secretory signal sequence (alsoknown as a leader sequence, prepro sequence or pre sequence) may beprovided in the recombinant vector. The secretory signal sequence isjoined to the DNA sequence encoding the polypeptide in the correctreading frame. Secretory signal sequences are commonly positioned 5′ tothe DNA sequence encoding the polypeptide. The secretory signal sequencemay be that normally associated with the polypeptide or may be from agene encoding another secreted protein.

[0075] For secretion from yeast cells, the secretory signal sequence mayencode any signal peptide which ensures efficient direction of theexpressed polypeptide into the secretory pathway of the cell. The signalpeptide may be naturally occurring signal peptide, or a functional partthereof, or it may be a synthetic peptide. Suitable signal peptides havebeen found to be the α-factor signal peptide (cf. U.S. Pat. No.4,870,008), the signal peptide of mouse salivary amylase (cf. O.Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modifiedcarboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987,pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or theyeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani etal., Yeast 6, 1990, pp. 127-137).

[0076] For efficient secretion in yeast, a sequence encoding a leaderpeptide may also be inserted downstream of the signal sequence andupstream of the DNA sequence encoding the polypeptide. The function ofthe leader peptide is to allow the expressed polypeptide to be directedfrom the endoplasmic reticulum to the Golgi apparatus and further to asecretory vesicle for secretion into the culture medium (i.e.exportation of the polypeptide across the cell wall or at least throughthe cellular membrane into the periplasmic space of the yeast cell). Theleader peptide may be the yeast α-factor leader (the use of which isdescribed in e.g. U.S. Pat. No. 4,546,082, EP 16 201, EP 123 294, EP 123544 and EP 163 529). Alternatively, the leader peptide may be asynthetic leader peptide, which is to say a leader peptide not found innature. Synthetic leader peptides may, for instance, be constructed asdescribed in WO 89/02463 or WO 92/11378.

[0077] For use in filamentous fungi, the signal peptide may convenientlybe derived from a gene encoding an Aspergillus sp. amylase orglucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, ora gene encoding a Humicola lanuginosa lipase. The signal peptide ispreferably derived from a gene encoding A. oryzae TAKA amylase, A. nigerneutral α-amylase, A. niger acid-stable amylase, or A. nigerglucoamylase.

[0078] For use in insect cells, the signal peptide may conveniently bederived from an insect gene (cf. WO 90/05783), such as the lepidopteranManduca sexta adipokinetic hormone precursor signal peptide (cf. U.S.Pat. No. 5,023,328).

[0079] The procedures used to ligate the DNA sequences coding for thepresent polypeptide, the promoter and optionally the terminator and/orsecretory signal sequence, respectively, and to insert them intosuitable vectors containing the information necessary for replication,are well known to persons skilled in the art (cf., for instance,Sambrook et al., op.cit.).

[0080] Host Cells

[0081] The DNA sequence encoding the present polypeptide introduced intothe host cell may be either homologous or heterologous to the host inquestion. If homologous to the host cell, i.e. produced by the host cellin nature, it will typically be operably connected to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment. The term“homologous” is intended to include a cDNA sequence encoding apolypeptide native to the host organism in question. The term“heterologous” is intended to include a DNA sequence not expressed bythe host cell in nature. Thus, the DNA sequence may be from anotherorganism, or it may be a synthetic sequence.

[0082] The host cell into which the DNA construct or the recombinantvector of the invention is introduced may be any cell which is capableof producing the present polypeptide and includes bacteria, yeast, fungiand higher eukaryotic cells.

[0083] Examples of bacterial host cells which, on cultivation, arecapable of producing the polypeptide of the invention are grampositivebacteria such as strains of Bacillus, such as strains of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces,such as S. lividans or S. murinus, or gramnegative bacteria such asEcherichia coli. The transformation of the bacteria may be effected byprotoplast transformation or by using competent cells in a manner knownper se (cf. Sambrook et al., supra).

[0084] When expressing the polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules (known as inclusion bodies), or may be directed to theperiplasmic space by a bacterial secretion sequence. In the former case,the cells are lysed and the granules are recovered and denatured afterwhich the polypeptide is refolded by diluting the denaturing agent. Inthe latter case, the polypeptide may be recovered from the periplasmicspace by disrupting the cells, e.g. by sonication or osmotic shock, torelease the contents of the periplasmic space and recovering thepolypeptide.

[0085] Examples of suitable mammalian cell lines are the COS (ATCC CRL1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCCCCL 61) cell lines. Methods of transfecting mammalian cells andexpressing DNA sequences introduced in the cells are described in e.g.Kaufmnan and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern andBerg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl.Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725;Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and vander Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982),841-845.

[0086] Examples of suitable yeasts cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae or Saccharomyces kluyveri. Methods for transforming yeastcells with heterologous DNA and producing heterologous polypeptidestherefrom are described, e.g. in U.S. Pat. No. 4,599,311, U.S. Pat. No.4,931,373, U.S. Pat. No. 4,870,008, U.S. Pat. No. 5,037,743, and U.S.Pat. No. 4,845,075, all of which are hereby incorporated by reference.Transformed cells are selected by a phenotype determined by a selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient, e.g. leucine. A preferred vector for use inyeast is the POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNAsequence encoding the polypeptide of the invention may be preceded by asignal sequence and optionally a leader sequence , e.g. as describedabove. Further examples of suitable yeast cells are strains ofKluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, orPichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132,1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).

[0087] Examples of other fungal cells are cells of filamentous fungi,e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichodermaspp., in particular strains of A. oryzae, A. nidulans or A. niger. Theuse of Aspergillus spp. for the expression of proteins is described in,e.g., EP 272 277 and EP 230 023. The transformation of F. oxysporum may,for instance, be carried out as described by Malardier et al., 1989,Gene 78: 147-156.

[0088] When a filamentous fungus is used as the host cell, it may betransformed with the DNA construct of the invention, conveniently byintegrating the DNA construct in the host chromosome to obtain arecombinant host cell. This integration is generally considered to be anadvantage as the DNA sequence is more likely to be stably maintained inthe cell. Integration of the DNA constructs into the host chromosome maybe performed according to conventional methods, e.g. by homologous orheterologous recombination.

[0089] Transformation of insect cells and production of heterologouspolypeptides therein may be performed as described in U.S. Pat. No.4,745,051; U.S. Pat. No. 4,879,236; U.S. Pat. Nos. 5,155,037; 5,162,222;EP 397,485; all of which are incorporated herein by reference. Theinsect cell line used as the host may suitably be a Lepidoptera cellline, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf.U.S. Pat. No. 5,077,214). Culture conditions may suitably be asdescribed in, for instance, WO 89/01029 or WO 89/01028, or any of theaforementioned references.

[0090] The transformed or transfected host cell described above is thencultured in a suitable nutrient medium under conditions permitting theexpression of the present polypeptide, after which the resultingpolypeptide is recovered from the culture.

[0091] The medium used for culturing the cells may be any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. in catalogues of the American Type Culture Collection).The polypeptide produced by the cells may then be recovered from theculture medium by conventional procedures including separating the hostcells from the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography,gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of polypeptide in question.

[0092] Transgenic Animals

[0093] It is also within the scope of the present invention to employtransgenic animal technology to produce the present polypeptide. Atransgenic animal is one in whose genome a heterologous DNA sequence hasbeen introduced. In particular, the polypeptide of the invention may beexpressed in the mammary glands of a non-human female mammal, inparticular one which is known to produce large quantities of milk.Examples of preferred mammals are livestock animals such as goats, sheepand cattle, although smaller mammals such as mice, rabbits or rats mayalso be employed.

[0094] The DNA sequence encoding the present polypeptide may beintroduced into the animal by any one of the methods previouslydescribed for the purpose. For instance, to obtain expression in amammary gland, a transcription promoter from a milk protein gene isused. Milk protein genes include the genes encoding casein (cf. U.S.Pat. No. 5,304,489), beta-lactoglobulin, alpha-lactalbumin and wheyacidic protein. The currently preferred promoter is thebeta-lactoglobulin promoter (cf. Whitelaw et al., Biochem J. 286, 1992,pp. 31-39). It is generally recognized in the art that DNA sequenceslacking introns are poorly expressed in transgenic animals in comparisonwith those containing introns (cf. Brinster et al., Proc. Natl. Acad.Sci. USA 85, 1988, pp. 836-840; Palmiter et al., Proc. Natl. Acad. Sci.USA 88, 1991, pp. 478-482; Whitelaw et al., Transgenic Res. 1, 1991, pp.3-13; WO 89/01343; WO 91/02318). For expression in transgenic animals,it is therefore preferred, whenever possible, to use genomic sequencescontaining all or some of the native introns of the gene encoding thepolypeptide of interest. It may also be preferred to include at leastsome introns from, e.g. the beta-lactoglobulin gene. One such region isa DNA segment which provides for intron splicing and RNA polyadenylationfrom the 3′ non-coding region of the ovine beta-lactogloblin gene. Whensubstituted for the native 3′ non-coding sequences of a gene, thissegment will enhance and stabilise expression levels of the polypeptideof interest. It may also be possible to replace the region surroundingthe initiation codon of the polypeptide of interest with correspondingsequences of a milk protein gene. Such replacement provides a putativetissue-specific initiation environment to enhance expression.

[0095] For expression of the present polypeptide in transgenic animals,a nucleotide sequence encoding the polypeptide is operably linked toadditional DNA sequences required for its expression to produceexpression units. Such additional sequences include a promoter asindicated above, as well as sequences providing for termination oftranscription and polyadenylation of mRNA. The expression unit furtherincludes a DNA sequence encoding a secretory signal sequence operablylinked to the sequence encoding the polypeptide. The secretory signalsequence may be one native to the polypeptide or may be that of anotherprotein such as a milk protein (cf. von Heijne et al., Nucl. Acids Res.14, 1986, pp. 4683-4690; and U.S. Pat. No. 4,873,316).

[0096] Construction of the expression unit for use in transgenic animalsmay conveniently be done by inserting a DNA sequence encoding thepresent polypeptide into a vector containing the additional DNAsequences, although the expression unit may be constructed byessentially any sequence of ligations. It is particularly convenient toprovide a vector containing a DNA sequence encoding a milk protein andto replace the coding region for the milk protein with a DNA sequencecoding for the present polypeptide, thereby creating a fusion whichincludes expression control sequences of the milk protein gene.

[0097] The expression unit is then introduced into fertilized ova orearly-stage embryos of the selected host species. Introduction ofheterologous DNA may be carried out in a number of ways, includingmicroinjection (cf. U.S. Pat. No. 4,873,191), retroviral infection (cf.Jaenisch, Science 240, 1988, pp. 1468-1474) or site-directed integrationusing embryonic stem cells (reviewed by Bradley et al., Bio/Technology10, 1992, pp. 534-539). The ova are then implanted into the oviducts oruteri of pseudopregnant females and allowed to develop to term.Offspring carrying the introduced DNA in their germ line can pass theDNA on to their progeny, allowing the development of transgenic herds.

[0098] General procedures for producing transgenic animals are known inthe art, cf. for instance, Hogan et al., Manipulating the Mouse Embryo:A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al.,Bio/Technology 6, 1988, pp. 179-183; Wall et al., Biol. Reprod. 32,1985, pp. 645-651; Buhler et al., Bio/Technology 8, 1990, pp. 140-143;Ebert et al., Bio/Technology 6: 179-183, 1988; Krimpenfort et al.,Bio/Tecnology 9: 844-847, 1991, Wall et al., J. Cell. Biochem. 49:113-120, 1992; U.S. Pat. No. 4,873,191, U.S. Pat. No. 4,873,316; WO88/00239, WO 90/05188; WO 92/11757 and GB 87/00458. Techniques forintroducing heterologous DNA sequences into mammals and their germ cellswere originally developed in the mouse. See, e.g. Gordon et al., Proc.Natl. Acad. Sci. USA 77: 7380-7384, 1980, Gordon and Ruddle, Science214: 1244-1246, 1981; Palmiter and Brinster, Cell 41: 343-345, 1985;Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985; andHogan et al. (ibid.). These techniques were subsequently adapted for usewith larger animals, including livestock species (see e.g., WO 88/00239,WO 90/01588 and WO 92/11757; and Simons et al., Bio/Technology 6:179-183, 1988). To summarize, in the most efficient route used to datein the generation of transgenic mice or livestock, several hundredlinear molecules of the DNA of interest are injected into one of thepro-nuclei of a fertilized egg according to techniques which have becomestandard in the art. Injection of DNA into the cytoplasm of a zygote canalso be employed.

[0099] Transgenic Plants

[0100] Production in transgenic plants may also be employed.

[0101] It has previously been described to introduce DNA sequences intoplants, which sequences code for protein products imparting to thetransformed plants certain desirable properties such as increasedresistance against pests, pathogens, herbicides or stress conditions(cf. for instance EP 90 033, EP 131 620, EP 205 518, EP 270 355, WO89/04371 or WO 90/02804), or an improved nutrient value of the plantproteins (cf. for instance EP 90 033, EP 205 518 or WO 89/04371).Furthermore, WO 89/12386 discloses the transformation of plant cellswith a gene coding for levansucrase or dextransucrase, regeneration ofthe plant (especially a tomato plant) from the cell resulting in fruitproducts with altered viscosity characteristics.

[0102] In the plant cell, the DNA sequence encoding the presentpolypeptide is under the control of a regulatory sequence which directsthe expression of the polypeptide from the DNA sequence in plant cellsand intact plants. The regulatory sequence may be either endogenous orheterologous to the host plant cell.

[0103] The regulatory sequence may comprise a promoter capable ofdirecting the transcription of the DNA sequence encoding the polypeptidein plants. Examples of promoters which may be used according to theinvention are the 35s RNA promoter from cauliflower mosaic virus CaMV),the class I patatin gene B 33 promoter, the ST-LS1 gene promoter,promoters conferring seed-specific expression, e.g. the phaseolinpromoter, or promoters which are activated on wounding, such as thepromoter of the proteinase inhibitor II gene or the wun1 or wun2 genes.

[0104] The promoter may be operably connected to an enhancer sequence,the purpose of which is to ensure increased transcription of the DNAsequence encoding the polypeptide. Examples of useful enhancer sequencesare enhancers from the 5′-upstream region of the 35s RNA of CaMV, the5′-upstream region of the ST-LS1 gene, the 5′-upstream region of the Cabgene from wheat, the 5′-upstream region of the 1′- and 2′-genes of theT_(R)-DNA of the Ti plasmid pTi ACH5, the 5′-upstream region of theoctopine synthase gene, the 5′-upstream region of the leghemoglobingene, etc.

[0105] The regulatory sequence may also comprise a terminator capable ofterminating the transcription of the DNA sequence encoding thepolypeptide in plants. Examples of suitable terminators are theterminator of the octopine synthase gene of the T-DNA of the Ti-plasmidpTiACH5 of Agrobacterium tumefaciens, of the gene 7 of the T-DNA of theTi plasmid pTiACH5, of the nopaline synthase gene, of the 35s RNA-codinggene from CaMV or from various plant genes, e.g. the ST-LS1 gene, theCab gene from wheat, class I and class II patatin genes, etc.

[0106] The DNA sequence encoding the polypeptide may also be operablyconnected to a DNA sequence encoding a leader peptide capable ofdirecting the transport of the expressed polypeptide to a specificcellular compartment (e.g. vacuoles) or to extracellular space. Examplesof suitable leader peptides are the leader peptide of proteinaseinhibitor II from potato, the leader peptide and an additional about 100amino acid fragments of patatin, or the transit peptide of variousnucleus-encoded proteins directed into chloroplasts (e.g. from theSt-LS1 gene, SS-Rubisco genes, etc.) or into mitochondria (e.g. from theADP/ATP translocator).

[0107] Furthermore, the DNA sequence encoding the polypeptide may bemodified in the 5′ non-translated region resulting in enhancedtranslation of the sequence. Such modifications may, for instance,result in removal of hairpin loops in RNA of the 5′ non-translatedregion. Translation enhancement may be provided by suitably modifyingthe omega sequence of tobacco mosaic virus or the leaders of other plantviruses (e.g. BMV, MSV) or of plant genes expressed at high levels (e.g.SS-Rubisco, class I patatin or proteinase inhibitor II genes frompotato).

[0108] The DNA sequence encoding the polypeptide may furthermore beconnected to a second DNA sequence encoding another polypeptide or afragment thereof in such a way that expression of said DNA sequencesresults in the production of a fusion protein. When the host cell is apotato plant cell, the second DNA sequence may, for instance, encodepatatin or a fragment thereof (such as a fragment of about 100 aminoacids).

[0109] The plant in which the DNA sequence coding for the polypeptide isintroduced may suitably be a dicotyledonous plant, examples of which areis a tobacco, potato, tomato, or leguminous (e.g. bean, pea, soy,alfalfa) plant. It is, however, contemplated that mono-cotyledonousplants, e.g. cereals, may equally well be transformed with the DNAsequence coding for the enzyme.

[0110] Procedures for the genetic manipulation of monocotyledonous anddicotyledonous plants are well known. In order to construct foreigngenes for their subsequent introduction into higher plants, numerouscloning vectors are available which generally contain a replicationsystem for E. coli and a selectable/screenable marker system permittingthe recognition of transformed cells. These vectors include e.g. pBR322,the pUC series, pACYC, M13 mp series etc. The foreign sequence may becloned into appropriate restriction sites. The recombinant plasmidobtained in this way may subsequently be used for the transformation ofE. coli. Transformed E. coli cells may be grown in an appropriatemedium, harvested and lysed. The chimeric plasmid may then be reisolatedand analyzed. Analysis of the recombinant plasmid may be performed bye.g. determination of the nucleotide sequence, restriction analysis,electrophoresis and other molecular-biochemical methods. After eachmanipulation the sequence may be cleaved and ligated to another DNAsequence. Each DNA sequence can be cloned on a separate plasmid DNA.Depending on the way used for transferring the foreign DNA into plantcells other DNA sequences might be of importance. In case the Ti-plasmidor the Ri plasmid of Agrobacterium tumefaciens or Agrobacteriumrhizogenes, at least the right border of the T-DNA may be used, andoften both the right and the left borders of the T-DNA of the Ri or Tiplasmid will be present flanking the DNA sequence to be transferred intoplant cells.

[0111] The use of the T-DNA for transferring foreign DNA into plantcells has been described extensively in the prior literature (cf. Gasserand Fraley, 1989, Science 244, 1293-1299 and references cited therein).After integration of the foreign DNA into the plant genome, thissequence is fairly stable at the original locus and is usually not lostin subsequent mitotic or meiotic divisions. As a general rule, aselectable marker gene will be cotransferred in addition to the gene tobe transferred, which marker renders the plant cell resistant to certainantibiotics, e.g. kanamycin, hygromycin, G418 etc. This marker permitsthe recognition of the transformed cells containing the DNA sequence tobe transferred compared to nontransformed cells.

[0112] Numerous techniques are available for the introduction of DNAinto a plant cell. Examples are the Agrobacterium mediated transfer, thefusion of protoplasts with liposomes containing the respective DNA,microinjection of foreign DNA, electroporation etc. In caseAgrobacterium mediated gene transfer is employed, the DNA to betransferred has to be present in special plasmids which are either ofthe intermediate type or the binary type. Due to the presence ofsequences homologous to T-DNA sequences, intermediate vectors mayintegrate into the Ri- or Ti-plasmid by homologous recombination. TheRi- or Ti-plasmid additionally contains the vir-region which isnecessary for the transfer of the foreign gene into plant cells.Intermediate vectors cannot replicate in Agrobacterium species and aretransferred into Agrobacterium by either direct transformation ormobilization by means of helper plasmids (conjugation). (Cf. Gasser andFraley, op. cit. and references cited therein).

[0113] Binary vectors may replicate in both Agrobacterium species and E.coli. They may contain a selectable marker and a poly-linker regionwhich to the left and right contains the border sequences of the T-DNAof Agrobacterium rhizogenes or Agrobacterium tumefaciens. Such vectorsmay be transformed directly into Agrobacterium species. TheAgrobacterium cell serving as the host cell has to contain a vir-regionon another plasmid. Additional T-DNA sequences may also be contained inthe Agrobacterium cell.

[0114] The Agrobacterium cell containing the DNA sequences to betransferred into plant cells either on a binary vector or in the form ofa cointegrate between the intermediate vector and the T-DNA region maythen be used for transforming plant cells. Usually either multicellularexplants (e.g. leaf discs, stem segments, roots), single cells(protoplasts) or cell suspensions are cocultivated with Agrobacteriumcells containing the DNA sequence to be transferred into plant cells.The plant cells treated with the Agrobacterium cells are then selectedfor the cotransferred resistance marker (e.g. kanamycin) andsubsequently regenerated to intact plants. These regenerated plants willthen be tested for the presence of the DNA sequences to be transferred.

[0115] If the DNA is transferred by e.g. electroporation ormicroinjection, no special requirements are needed to effecttransformation. Simple plasmids e.g. of the pUC series may be used totransform plant cells. Regenerated transgenic plants may be grownnormally in a greenhouse or under other conditions. They should displaya new phenotype (e.g. production of new proteins) due to the transfer ofthe foreign gene(s). The transgenic plants may be crossed with otherplants which may either be wild-type or transgenic plants transformedwith the same or another DNA sequence. Seeds obtained from transgenicplants should be tested to assure that the new genetic trait isinherited in a stable Mendelian fashion.

[0116] See also Hiatt, Nature 344: 469-479, 1990; Edelbaum et al., J.Interferon Res. 12: 449-453, 1992; Sijmons et al., Bio/Tecnology 8:217-221, 1990: and EP 255 378.

[0117] Uses

[0118] In the pharmaceutical composition of the invention, the presentpolypeptide may be formulated by any of the established methods offormulating pharmaceutical compositions, e.g. as described inRemington's Pharmaceutical Sciences, 19 th. edition,1995. Thecomposition may be in a form suited for systemic injection or infusionand may, as such, be formulated with sterile water or an isotonic salineor glucose solution. The compositions may be sterilized by conventionalsterilization techniques which are well known in the art. The resultingaqueous solutions may be packaged for use or filtered under asepticconditions and lyophilized, the lyophilized preparation being combinedwith the sterile aqueous solution prior to administration. Thecomposition may contain pharmaceutically acceptable auxiliary substancesas required to approximate physiological conditions, such as bufferingagents, tonicity adjusting agents and the like, for instance sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc.

[0119] The pharmaceutical composition of the present invention may alsobe adapted for oral, nasal, transdermal, transepithelial or rectaladministration. The pharmaceutically acceptable carrier or diluentemployed in the composition may be any conventional solid carrier.Examples of solid carriers are lactose, terra alba, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.Similarly, the carrier or diluent may include any sustained releasematerial known in the art, such as glyceryl monostearate or glyceryldistearate, alone or mixed with a wax. For oral administration, thecomposition may be tabletted, placed in a hard gelatin capsule in powderor pellet form or it can be in the form of a troche or lozenge. Theamount of solid carrier will vary widely but will usually be from about25 mg to about 1 g. The present polypeptide may also be placed in a softgelatin capsule in a liquid carrier such as syrup, peanut oil, olive oilor water.

[0120] The polypeptides of the invention are effective over a widedosage range. A typical dosage is in the range of from 0.05 to about1000 mg, preferably from about 0.1 to about 500 mg, and more preferredfrom about 0.5 mg to about 200 mg per day administered in one or moredosages such as 1 to 3 dosages. The exact dosage will depend upon thefrequency and mode of administration, the sex, age, weight and generalcondition of the subject treated, the nature and severity of thecondition treated and any concomitant diseases to be treated as well asother factors evident to those skilled in the art.

[0121] The polypeptide of the invention is contemplated to beadvantageous for use in therapeutic applications within appetitesuppression or satiety induction, such as for the prophylaxis ortreatment of diseases or disorders associated with impaired appetiteregulation. Examples of such diseases or disorders are obesity, type IIdiabetes and bulimia. The dosage of the polypeptide administered to apatient will vary with the type and severity of the condition to betreated, but is generally in the range of 0.01-5.0 mg/kg body weight perday in one or more dosages such as 1 to 3 dosages.

[0122] Furthermore, the polypeptide of the invention is contemplated tobe advantageous for treatment of autoimmune disorders, inflammation,arthritis, type I diabetes, multiple schlerosis, stroke, osteoporosis,septic shock, symptoms of menopause, menstrual complications andParkinson's disease.

[0123] The present invention is further illustrated by the followingrepresentative examples which are, however, not intended to limit thescope of the invention in any way. Fur further details of the inventionreference should also be made to Peter Kristensen et al., “HypothalamicCART: a novel anorectic peptide regulated by leptin”, Nature, in press,which is hereby incorporated by reference.

EXAMPLES Example 1

[0124] Isolation of CART

[0125] Total RNA from cultivated cells were prepared from primary celllines derived from the rat tumors MSL-A-AN and MSL-A-M3 (Madsen et al,Scand. J. Clin. Invest. Supplement 220: 27-36) by the method ofChomczynski & Sacchi (1987). From this we made poly-A mRNA usingPharmacia's “Quick Prep® micro” mRNA purification kit. Double strandedcDNA was made using Clontech's “cDNA clone I” synthesis kit. Eco RIadaptors CA6/CA7 (Ace et al. (1994)) were added to the blunt endedcDNA's.

[0126] 50 ng of each cDNA was amplified using the CA6 primer. The primerused for amplification of the MSL-A-M3 cDNA was biotinylated (thedriver). The amplified MSL-A-AN cDNA was cut with Eco RI (the tracer).

[0127] 8 μg of the biotinylated M3 cDNA was bound to 60 μl of DYNALmagnetic streptavidin beads, treated with NaOH, washed, 2×hybridizationbuffer was added, and the mixture was heated to 68° C. 0.5 μg of the EcoRI cut AN cDNA was heated to 98° C. and added to the tube with themagnetic beads. The reaction tube was incubated at 68° C. for 20 hours.The magnetic beads with the bound cDNA and the tracer which hadhybridized to it was removed using a magnet.

[0128] This procedure was repeated three times, the last two with 10 μgof driver DNA and 80 μl of magnetic beads.

[0129] The remaining tracer DNA was purified on a Chromaspin 100 columnfrom Clontech and cloned into an Eco RI cut vector (pCR™II, Invitrogen)and transformed into E. coli (ElectroMax).

[0130] The cells were plated on LB plates with 100 μg/ml Ampicillin. Theplates were replicated and the cells from the replica were transferredto nylon filters (Hybond-N+, Amersham). The filters were hybridized withradioactively labelled driver cDNA and autoradiographs were made. Thefilters were then stripped for the radioactive probe and thereafter theprocedure was repeated with a probe made from the tracer cDNA.

[0131] The autoradiographs were compared and spots that were onlypresent with the latter probe were identified and the correspondingcolonies were isolated.

[0132] DNA sequencing of the inserted DNA in one of these colonies wereidentified as CART (Cocaine and Amphetamine Regulated Transcript fromrat brain (Douglass et al. (1995)). This transcript codes for a protein(polypeptide) of 129 or 116 amino acids (differential splicing of an inframe 39 bp intron). The polypeptide seems to have a signal sequence inthe amino terminal end, and the secreted part contains several dibasicamino acid pairs which could be “pro hormone” processing sites.

Example 2

[0133] Cloning of Rat CART

[0134] In order to clone the whole coding region of the CART geneprimers were made that overlaps with the start codon and with the stopcodon, respectivly.

[0135] MHJ4754: 5′-AAAAAGGATCCACCATGGAGAGCTCCCGCC-3′

[0136] Bold: Bam HI site for cloning. Underlined: ATG start codon.

[0137] MHJ4753: 5′-AAAAAAGCTTCACAAGCACTTCAAGAGGAAA-3′

[0138] Bold: Hin dIII site for cloning. Underlined: TGA stop codon,opposite strand.

[0139] As template for the PCR cloning we used the same double strandedcDNA preparation as described in Example 1 (from MSL-A-AN). The PCRreaction, 25 cycles: 60 sec 94° C. 30 sec 52° C. 60 sec 72° C.

[0140] Two bands appeared when the reaction mix was run on a 2% agarosegel corresponding to the two splice variants mentioned in Douglass etal. (1995).

[0141] Each of the two bands were isolated, cut with Bam HI and HindIII, and cloned into Bam HI-Hin dIII cut pSX221 (fragments A,B,C,D, andE ligated into pSX191, WO 92/11357) giving rise to pSX592 and pSX593(short and long form, respectively) corresponding to SEQ ID Nos. 2 and1, respectively (see FIG. 1).

Example 3

[0142] Expression of Rat CART in E. coli I

[0143] In order to express CART in E. coli three constructs were madewhere different forms of CART were fused to Glutathione S-transferaseusing the pGEX system from Pharmacia P-L Biochemicals.

[0144] The different forms of CART, full length of both splice variants(starting with Gln-Glu-Asp) and the form starting with Ile-Pro-Ile(Spiess et al. (1981)) were amplified using PCR primers that add a BamHI site (bold) and the four triplets that codes for the Factor Xaprotease recognition site Ile-Glu-Gly-Arg in the 5′-end (N-terminus). As3′-end primer we used the same as in Example 2 (MHJ4753). As templateswere used the plasmids pSX592 and pSX593 described in Example 2.MHJ4885: 5′-AAAAAGGATCC ATC GAA GGT CGT CAG GAG GAT GCC GAG CTG-3′               Ile Glu Gly Arg Gln Glu Asp Ala Ser Leu MHJ4880:5′-AAAAAGGATCC ATC GAA GGT CGT ATT CCG ATC TAT GAG AAG A-3′               Ile Glu Gly Arg Ile Pro Ile Tyr Glu Lys

[0145] The PCR reaction, 25 cycles: 60 sec 94° C. 30 sec 55° C. 60 sec72° C.

[0146] The reaction mixtures were cut with Hin dIII, filled out withKlenow polymerase, and then cut with Bam HI. They were then run on a 2%agarose gel and the bands corresponding to the three variants wereisolated and cloned into pGEX-2T (cut with Eco RI, filled out, and thencut with Bam HI) giving rise to pSX600 (IPI-CART) corresponding to SEQID No. 4, pSX 601 (short form) corresponding to SEQ ID No. 2, and pSX605(long form) corresponding to SEQ ID No. 1 (see FIG. 2).

Example 4

[0147] Expression of Rat CART in E. coli II

[0148] In order to express CART in E. coli three constructs were madewhere different forms of CART were fused to Thioredoxin using theThioFusion™ Expression System from Invitrogen Corporation.

[0149] The different forms of CART, full length of both splice variants(starting with Gln-Glu-Asp) and the form starting with Ile-Pro-Ile(Spiess et al. (1981)) were amplified using PCR primers that add a BamHI site (bold) and the four triplets that codes for the Factor Xaprotease recognition site Ile-Glu-Gly-Arg in the 5′-end (N-terminus) anda Hin dIII (bold) site in the 3′-end. As templates were used the pGEXfusion constructs described in Example 3. MHJ5141: 540 -AAAAAGGATCCG ATCGAA GGT CGT GAG GAG GAT-3′                 Ile Glu Gly Arg Gln Glu AspMHJ5140: 540 -AAAAAGGATCCG ATG GAA GGT CGT ATT CGG ATC-3′                Ile Glu Gly Arg Ile Pro Ile MHJ5142: 5′-AAAAAGTCGATAAGCTTCACAAGCACTTCAAGAGGA-3′

[0150] Bold: Hin dIII Underlined: Stop codon on opposite strand The PCRreaction, 25 cycles: 60 sec 94° C. 30 sec 52° C. 60 sec 72° C.

[0151] The reaction mixtures were cut with Hin dIII, filled out withKlenow polymerase and then cut with Bam HI. They were then run on a 2%agarose gel and the bands corresponding to the three variants wereisolated and cloned into pTrxFus (cut with Sal I, filled out, and thencut with Bam HI) giving rise to pSX610 (long form) corresponding to SEQID No. 1, pSX611 (short form) corresponding to SEQ ID No. 2, and pSX612(IPI-CART) corresponding to SEQ ID No. 4 (see FIG. 3).

[0152] The plasmids were transformed into E. Coli G1724 (Invitrogen) andthe the resulting strains were cultivated according to the manual forthe ThioFusion™ Expression System kit.

[0153] The fusion proteins were purified according to the instructionmanual for ThioBond™ Resin (Invitrogen Corporation). The purified fusionproteins were then treated with the endoproteinase Factor Xa (BoehringerMannheim). Ratio Factor Xa/Fusion protein=1/800. Incubation: 4° C., 16hours.

Example 5

[0154] Expression of Rat CART in S. cerevisiae

[0155] The Yeast-E.coli shuttle vector used in the following example(pAK405) contains a heterologous protein expression cassette, whichincludes a DNA sequence encoding a modified MFα1 leader sequence (with aNcoI site added in the 3′-end) followed by the heterologous polypeptidein question operably placed in between the TPI promoter and the TPIterminator of S. cerevisiae in a POT plasmid (Kjeldsen et al., Gene170:107-112, 1996).

[0156] Two primers CART1 and CART2 were constructed. These allow a PCRproduct to be made that furnishes the DNA sequence encoding either theshort or the long form of full length CART with a 5′ NcoI site and a 3′XbaI site allowing insertion into the yeast-E.coli shuttle vectorpAK405.                      NcoI site                      ˜˜˜˜˜˜˜˜˜CART1: 5′-ACG GAG GAG CCC ATG GCT AAG AGA CAG GAG GAT GCC GAG CTG CAGC-3′                      XbaI site                      ˜˜˜˜˜˜˜˜˜CART2: 5′-CTT AAC GGC TTC TAG ATC ACA AGC ACT TCA AGA GG-3′

[0157] The following Polymerase Chain Reaction (PCR) was performed usingthe Pwo DNA polymerase (Boehringer Mannheim) according to themanufacturers instructions.

[0158] 5 μl primer CART1 (100 pmol)

[0159] 5 μl primer CART2 (100 pmol)

[0160] 10 μl 10×PCR buffer+MgSO₄

[0161] 8 μl dNTP mix

[0162] 0.5 μl Pwo enzyme

[0163] 1 μl pSX592 or pSX593 plasmid as template (0.2 μg DNA)

[0164] 70.5 μl H₂O

[0165] A total of 16 cycles was performed. One cycle was as follows: 45sec at 94° C., 1 min at 42° C., and 1.5 min at 72° C.

[0166] The resulting PCR products were cut with restriction enzymes NcoIand XbaI and ligated with the BstXI/XbaI fragment and the BstXI/NcoIfragment of pAK405. BstXI, NcoI and XbaI cutat positions 701, 1419 and1616 of pAK405, respectively. The construction and DNA sequence of theresulting heterologous expression cassettes are shown in FIGS. 4 and 5,respectively.

[0167] The resulting plasmids pEA182 (short form of CART) and pEA183(long form of CART) were transformed into S. cerevisia strain ME1487(MATα Δyap3::URA3 pep4-3 Δtpi::LEU2 leu2 HIS4 ΔURA3, described in patentapplication DK 0749/96). Transformants were selected by glucoseutilization as a carbon source in YPD plates (1% w/v yeast extract, 2%w/v peptone, 2% glucose, 2% agar). yEA182 corresponding to SEQ ID No. 2and yEA183 corresponding to SEQ ID No. 1 are the transformants obtainedfrom the plasmids pEA182 and pEA183, respectively.

[0168] A similar construct was made which produces IPI-CART: pSX630corresponding to SEQ ID No. 4.

[0169] Transformants were cultivated in YPD liquid medium at 30° C. for3 days with shaking at 200 rpm. Culture supernatants were obtained aftercentrifugation and supernatants were analysed for CART related material.

[0170] Human CART(1-89) corresponding to SEQ ID No. 3 differs from therat form by having a valine residue in position 42 in stead of aisoleucine residue. Human CART(1-89) may be prepared in analogy with theabove examples starting from a human tissue or simply by substitutingvaline for isoleucine in position 42 of rat CART(1-89) according tomethods well-known to a person skilled in the art.

Example 6

[0171] Preparation of Rat CART(68-102, Long). SEQ ID No. 9

[0172] Ten mg of rat CART(54-102, long), SEQ ID No.6, prepared asdescribed in Example 7, was dissolved in 2 ml of 70% (v/v) formic acid.A crystal corresponding to approx. 1 mg of cyanogenebromide was added tothe dissolved peptide and the mixture was allowed to stand dark at roomtemperature for 16 hours. The generated CART fragment, CART(68-102,long) was purified by preparative HPLC as described in Example 7.

Example 7

[0173]E.Coli Construction

[0174] The thioredoxin-CART short form fusion protein was isolated from2 litres of E.Coli fermentation broth and subjected to FXa cleavage.

[0175] This digest mixture was analysed by HPLC (FIG. 6). Fractionscorresponding to the two main peaks (Fr. 15 and Fr. 28, FIG. 6) weresubjected to sequence and mass spectrometry analysis: Fr. No. Sequencefound Mass found Theoretical mass 15 ALDIYSAVDD . . .  8882.8  8882.4 28SDKIIHLTDD . . . 13529.0 13529.5

[0176] The pept

[0177] ide found in Fraction 15 is identical to rat CART(10-89)corresponding to SEQ ID No. 10, whereas the peptide in Fraction 28 isthe thioredoxin split product with the C-terminal sequence of . . .IEGR. The small peptide fragment, CART(1-9), was not identified in thedigest.

[0178] From 2.1 of fermentation broth the total of 4.0 mg of CART(10-89)was isolated.

[0179] Human CART(10-89) corresponding to SEQ ID No. 11 differs from therat form by having a valine residue in position 33 in stead of aisoleucine residue. Human CART(10-89) may be prepared in analogy withthe above example starting from a human tissue or simply by substitutingvaline for isoleucine in position 33 of rat CART(10-89) according tomethods well-known to a person skilled in the art.

[0180] Yeast Construction

[0181] The fermentation broth from the 5 litres yeast fermentation(yEA183, long form) was analysed by HPLC (FIG. 7). A series ofexpression products was seen in this analysis.

[0182] Preliminary sequence analysis indicated that several of thepeptides eluting at a retention time between 20 and 30 min. werefragments of the mature full length CART molecule. The total amount ofCART related products in the fermentation broth was approx. 250mg/litre.

[0183] The CART fragments from 4.25 litres of fermentation broth wereseparated using the following method:

[0184] The fermentation broth (pH=4.6, Λ=8 mS) was adjusted to pH=5.0and diluted with 25 litres of water (resulting Λ=1.3 mS) and pumped (500ml/h) onto a SP-Sepharose column (5×15 cm) previously equilibrated with50 mM HAc at pH=5.0. The column was eluted with a linear gradientbetween 1500 ml of 50 mM HAc and 1500 ml of 50 mM HAc containing 1.0 MNaCl. Fractions of 10 ml were collected and analysed for the content ofCART fragments by analytical HPLC. The chromatogram from the ionexchange chromatography is shown in FIG. 8. Three pools (A, B and C, seeFIG. 8) were generated on the basis of the analytical HPLC analysis ofthe individual fractions. Each of these pools, representing a welldefined CART fragment, were further purified by preparative HPLC. Theindividual pools (120-150 ml) were pumped on a Vydac 214TP1022 (100 ml)reverse phase C4 HPLC column previously equilibrated with 0. 1% TFA. Thecolumn was washed with 100 ml 0.1% TFA and eluted with a linear gradientfrom 0 to 70% MeCN in 0.1% TFA at a flow rate of 3 ml/min. Theindividual fractions from the 3 preparative HPLC purifications wereanalysed by analytical HPLC and the 3 individual CART fragments wereisolated from these fractions by lyophilisation.

[0185] Characterisation of the Isolated CART Fragments From the YeastFermentation

[0186] The purity of the 3 isolated CART fragments are shown in FIGS. 9,10, and 11, respectively. The structure of the 3 purified CART fragmentswere determined by amino acid sequencing and MALDI-TOF massspectrometry. The following results were found: Pool Mass TheoreticalNo. Sequence found found mass Identity A YGQVPM . . . 4389.9 4387.1CART(62-102) B KYGQVP . . . 4516.5 4515.3 CART(61-102) C RIPIYEKKY . . .5418.0 5415.4 CART(54-102)

[0187] The total yields of the purified rat CART fragments from 4.25litres of fermentation broth were: Pool No. Identity Total Yield ACART(62-102)  33 mg B CART(61-102) 200 mg C CART(54-102) 280 mg

[0188] CART(62-102) corresponds to SEQ ID No. 8, CART(61-102)corresponds to SEQ ID No. 7 and CART(54-102) corresponds to SEQ ID No.6.

[0189] The human CART(62-102) and CART(61-102) fragments are identicalto the rat fragments. Human CART(54-102) differs from the rat fragmentby having a valine residue in position 2 in stead of a isoleucineresidue. Human CART(54-102) may be prepared using the same method asdescribed for rat CART(54-102) starting from a human tissue or simply bysubstituting valine for isoleucine in position 2 of rat CART(54-102)according to methods well-known to a person skilled in the art.

Example 8

[0190] The Disulphide Bond Configuration in Rat CART(62-102) SEQ ID No.8

[0191] The C-terminal part of the CART molecule contains 6 cysteineresidues: Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe- Leu-Leu-Lys-Cys-Leu

[0192] In principle these 6 cysteine residues can exist in 5×3×1=15possible arrangements to form 3 disulphide bonds. The present series ofexperiments were carried out in order to elucidate, which of the 15possible arrangements was present in the CART molecule.

[0193] The CART fragment (residues 62-102) prepared as described in thepreceding example was digested with Armillaria Mellea protease, whichcleaves specifically on the N-terminal side of lysine residues. Thefragments generated were separated by HPLC and subjected to massspectrometry and amino acid sequence analyses. From mass spectrometryand sequence analysis it could be deduced that the following twofragments were generated by the Armillaria Mellea protease digestion:Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-ArgLys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-LeuLys-Cys-Leu Lys-Gly-Ala-Arg-Ile-Gly

[0194] The first of these fragments is a 3 chained molecule still heldtogether by the 3 disulphide bonds. This molecule was subjected todigestion with Pseudomonas fragi endoproteinase Asp-N, which cleavesspecifically on the N-terminal side of aspartic acid residues. From thisdigest the following two fragments could be isolated:Tyr-Gly-Gln-Val-Pro-Met-Cys Lys-Leu-CysAsp-Ala-Gly-Glu-Gln-Cys-Ala-Val-ArgAsp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu- Leu Lys-Cys-Leu

[0195] The first of these fragments is a two chained molecule heldtogether by a single disulphide bond. Thus, cysteine residue I and IIIof the original molecule must be linked.

[0196] The second of these fragments, containing cysteine residue II,IV, V and VI, is a 3 chained molecule linked by 2 disulphide bonds. Thismolecule was subjected to trypsin digestion and the following twofragments were generated: Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-ArgGly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu Asp-Cys-Pro-Arg Lys-Cys-Leu

[0197] From these results it is clear that Cys-II and Cys-V are linkedand that Cys-IV and Cys-VI are linked.

[0198] From the above results the entire primary and secondary structureof the C-terminal part of the CART molecule can be deduced showing thatthe disulphide bond exists in a I-III, II-V and IV-VI configuration (seeFIG. 12).

Example 9

[0199] Test Method for Measuring Appetite Suppression in Mice

[0200] Mice were deprived of their normal feed for two days and givenfree access to a solution of nutritionally complete infant formula milk(Complan®) for the first day, after which food deprivation was completefor the last day before testing. After one day of food deprivation, micewere injected intra-cerebroventricularly (ICV) in the lateral ventriclewith 10 microlitres of a solution containing the test substance. Thirtyminutes after injection, mice were individually placed in a 15×15×15 cmtest box with a stainless steel grid floor and a glass drinking tubewhich projected into the box. The drinking tube was connected to areservoir containing the formula milk solution, and the interior of thedrinking tube contained an electrode enabling the detection of drinkingcontacts with the solution by measuring the flow of a weak(unnoticeable) electric current through mice by means of an electronicapparatus connected to the drinking tube electrode and the stainlesssteel grid floor. Consumption of the milk solution was measured over a10 minutes period by electronically recording the total amount ofcontact with the milk solution during the test session. The degree ofappetite suppression produced by a given test substance was determinedby statistical comparison of the duration of milk consumption by control(vehicle treated) mice with that of mice treated with a test substance.The degree of appetite suppression in a treated group of mice wasexpressed as percent reduction of consumption relative to the mean ofthe control group's response.

Example 10

[0201] Test for Appetite Suppression in Mice by Recombinant RatCART(10-89. Short), SEQ ID No. 10

[0202] Mice were tested for appetite suppression as described in Example9 after treatment with 1-20 micrograms of a test substance consisting ofrecombinant rat CART(10-89) dissolved in phosphate buffered saline.Intra-cerebroventricular injections of the test substance producedstatistically significant suppression of milk consumption. Dose(micrograms) 1 2 5 10 20 % Feeding Suppression 41% 70% 71% 59% 78%

Example 11

[0203] Test for Appetite Suppression in Mice by Recombinant RatCART(54-102, Long), SEQ ID No. 6

[0204] Mice were tested for appetite suppression as described in Example9 after treatment with 0.5-10 micrograms of a test substance consistingof recombinant rat CART(54-102) dissolved in phosphate buffered saline.Intra-cerebroventricular injections of the test substance producedStatistically significant suppression of milk consumption. Dose(micrograms) 0.5 1 2 5 10 % Feeding Suppression 13% 71% 100% 100% 100%

Example 12

[0205] Test for Appetite Suppression in Mice by Recombinant RatCART(61-102. Long). SEQ ID No. 7

[0206] Mice were tested for appetite suppression as described in Example9 after treatment with 0.5-10 micrograms of a test substance consistingof recombinant rat CART(61-102) dissolved in phosphate buffered saline.Intra-cerebroventricular injections of the test substance producedstatistically significant suppression of milk consumption. Dose(micrograms) 0.5 1 2 5 10 % Feeding Suppression 74% 80% 78% 100% 100%

Example 13

[0207] Test for Appetite Suppression in Mice by Recombinant RatCART(62-102, Long), SEQ ID No. 8

[0208] Mice were tested for appetite suppression as described in Example9 after treatment with 0.5-10 micrograms of a test substance consistingof recombinant rat CART(62-102) dissolved in phosphate buffered saline.Intra-cerebroventricular injections of the test substance producedstatistically significant suppression of milk consumption. Dose(micrograms) 0.5 1 2 5 10 % Feeding Suppression 30% 55% 60% 89% 100%

Example 14

[0209] Effect of Fasting on the Expression of CART mRNA

[0210] Rat brain tissue from three different groups of animals (6animals in each group): normal control, fasted for 48 hours and fastedfor 48 hours and re-fed for 3 hours. Cryostat sections Were cut andthree sections were used for in situ hybridisation with 35-S labelledanti-sense CART RNA. Additional sections were included with a similaramount of 35-S labelled sense RNA probe. Slides were exposed on oneBetamax hyperfilm for 7 days. The images were digitized and analysedusing the NIH Image software (treatment blinded to the observer). Anempirically determined gray level was used to set the threshold on allimages after exclusion of those with bad morphology or badrepresentation of the area in question (indicated by lines on thefigure). The average gray scale value of all pixels above this levelwithin the area of interest (PVN or Nucleus Arcuatus (in both thefrontal section and at eminentia mediana)) was then measured andmultiplied by the size of the area of interest. A mean for each animalwas then determined and the standard deviation indicated represents thespreading between animals.

[0211] These results show that CART mRNA is regulated in a mannerinverse to that of NPY thus indicating the presence of neurotransmittermode action for CART involving a receptor mediating a satiety stimulus,presumably along the arcuate—paraventricular nucleus pathway (see FIG.13).

Example 15

[0212] Low Expression of CART mRNA in Arcuate Nucleus of Obese ZuckerRats

[0213] Rat brain tissue was obtained from two groups of Zucker rats (6animals in each group): 25 fa/fa and fa/+. Cryostat sections were cutand three sections were used for in situ hybridisation with 35-Slabelled anti-sense CART RNA (rCART5A cDNA (Eco47-HindIII fragment frombp Nos. 226-411)). Post-hybridisation washings were performed at 57° C.and 62° C. in 50% formamide. Additional sections were included with asimilar amount of 35-S labelled sense RNA probe and these showed nosignal. Slides were exposed on one Betamax hyperfilm for 12 days. Theimages were digitized to 256 grey levels and analysed using the NIHImage software (treatment blinded to the observer). An empiricallydetermined gray level (100) was used to set the threshold on all imagesafter exclusion of those with bad morphology or bad representation ofthe area in question (three sections) and one set of slides (Nos. 25-27)as one animal due to an error was represented twice (and one missing).The average gray scale value of all pixels above the arcuate nucleus wasthen measured. A mean for each animal was then determined and theproduct of the area and mean calculated. These results show that CARTmRNA is regulated in a manner inverse to that of NPY thus indicating thepresence of neurotransmitter mode action for CART involving a receptormediating a sateity stimulus, presumably along thearcuate—paraventricular nucleus pathway. Furthermore, the strongdecrease in CART expression in the obese Zucker rat deficient in leptinsignalling strongly implicates CART mediated neuronal signalling in asateity mediating pathway in the hypothalamus (see FIG. 14).

Example 16

[0214] Preparation of Rat CART(55-102). SEQ ID No. 4

[0215] Plasmid pSX637, encoding Glu-Glu-Ile-Asp-CART(55-102), wasconstructed by the use of the PCR technique “Splicing by OverlapExtension” (Horton et al., Gene 77:61-68, 1989) and the product wasinserted into pAK405 (Example 5). The resulting expression plasmid(pSX637) is shown in FIG. 15. As can be seen from this figure a sequenceof: Lys-Glu-Leu-Glu has been placed between the α-leader and Kex2 sitein order to optimise processing (Kjeldsen et al., Gene 170:107-112,1996).

[0216] Plasmid pSX637 was transformed into Saccharomyces. cerevisiaestrains ME1487 (MATα Δyap3::URA3 Δtpi::LEU2 pep4-3 Δura3 leu2) andME1719 (MATα/MATα Δyap3::URA3/Δyap3::URA3 Δtpi::LEU2/Δtpi::LEU2pep4-3/pep4-3 Δura3/Δura3 leu2/leu2), respectively. Host cells werecultured in YPGGE medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2%(w/v) galactose, 2% (v/v) glycerol and 1 % (v/v) ethanol) to OD_(600 nm)of 0.2. Transformation was made by using a standard protoplast method.Transformant YES1789, was obtained containing the EEID-CART expressingplasmid after transformants were selected on minimal plates containingglucose.

[0217] Fermentation of EEID-CART(55-102) (yeast strain: YES1789) wascarried out in 6 L stainless steel fermentor from Chemap A/B,Switzerland equipped with bottom stirrer and in situ steamsterilization. The medium was composed essentially as previouslydescribed (Thim et al., FEBS Lett. 318:345-352, 1993) and a startingvolume of 4.0 L was chosen. Ammonia was added to adjust pH throughoutthe fermentation to 4.9 and the temperature was kept constant at 30° C.with steam/cooling water. Dissolved oxygen was kept above 50% saturationby frequent adjustment of the stirrer speed. The inoculum was from a YPD(Yeast-extract Peptone Dextrose) culture (2 days, 30° C.). Glucose (1250g) was dissolved in water to a volume of 2 L, sterilised separately inan autoclave (30 min, 121 ° C.), and added with a constant rate of 30g/h over the first 24 hours. The rate was increased to 60 g/h over thenext 24 hours. After 48 hours of cultivation the broth was harvested.

[0218] The EEID-CART(55-102) broth was adjusted to pH 11 with 3 N NaOHand kept at 25° C. for 30 min before centrifugation as above. Thesupernatant was adjusted to pH 9.7 to protect against proteolysis beforethe purification was initiated. The dry biomass in the two fermentationswas 73.6 g/L. The weight of the total fermentation broth was 5706 g. Thefermentation supernatant (4.6 L) from yeast strain YES1789 expressingGlu-Glu-Ile-Asp-CART(55-102) was dialysed against 60 L of water at 4° C.for 96 h. The pH was adjusted to 4.3 and the solution was pumped onto aSP-Sepharose (Pharmacia) column (5×15 cm) with a flow rate of 300 mL/h.Prior to the application the column was equilibrated with 50 mM HAcbuffer pH 4.25. The column was washed with 3 L of 50 mM HAc buffer pH4.25. EEID-CART(55-102) was eluted from the column by a linear gradientbetween 1.5 L of 50 mM HAc buffer pH 4.25 and 1.5 L of 50 mM HAc bufferpH 4.25 containing 1M NaCl. Fractions (10 mL) were collected at a flowrate of 100 mL/h and the absorbance was measured at 280 nm. TheEEID-CART(55-102) molecule eluted at 0.5M of NaCl and fractionscontaining the peptide were dialysed against 25 L of 50 mM HAc buffer pH4.5 at 4° C. for 96 h. L-Cystein was added to the solution (560 mL) togive a final concentration of 1 mM, and 4.5 mLdipeptidylaminopeptidase-1 ( DAP-1, Cathepsin C from chicken liver, EC3.4.14.1, Unizyme Laboratories) was added. The resulting concentrationof DAP-1 was 20 units/mL. The digestion of EEID-CART(55-102) was carriedout at 37° C. and aliquots were analysed by HPLC each half hour. Afterincubation for 4.5 h more than 98% of the precursor was converted toCART(55-102). The digestion mixture was adjusted to pH 4.25 and pumped(60,mL/h) onto a SP-Sepharose (Pharmacia) column (5×8 cm) previouslyequilibrated with 50 mM HAc buffer pH 4.25. The column was washed with1.6 L of equilibration buffer and CART(55-102) was eluted with a lineargradient between 1 L of 50 mM HAc buffer pH 4.25 and 1 L of 50 mM HAcbuffer pH 4.25 containing 1.2 M NaCl, at a flow rate of 100 mL/h. Theabsorbance at 280 nm was recorded and fractions of 10 mL were collected.The CART(55-102) molecule eluted at 0.67 M of NaCl and fractionscontaining the peptide were pooled. The solution was divided into 5equal portions. Each portion was pumped on a Vydac 214TP1022 reversephase C4 preparative HPLC column (2.2×25 cm) previously equilibratedwith 0.1% (v/v) TFA. The column was washed with 100 mL 0.1% (v/v) TFAand eluted with a linear gradient from 0 to 70% (v/v) acetonitrile in0.1% (v/v) TFA at a flow rate of 3 mL/min. The CART(55-102) containingfractions from the 5 preparative HPLC purifications were pooled and thepeptide was isolated from these fractions by lyophilisation. The totalyield of CART(55-102) from 4.6 L of fermentation supernatant was 705 mg.

[0219] N-terminal amino acid sequences were determined by automatedEdman degradations using an Applied Biosystem Model 494 ProteinSequencer essentially as described by the manufacturer. The N-terminalsequence of the purified CART(55-102) was found to be:

[0220] IPIYEKKYGQ . . .

[0221] Mass spectrometric analysis on the isolated CART(55-102) wasperformed on a Voyager RP MALDI-TOF instrument (Perseptive BiosystemsInc., Framingham, Mass.) equipped with a nitrogen laser (337 nm). Theinstrument was operated in linear mode with delayed extraction, and theaccelerating voltage in the ion source was 25 kV.

[0222] Sample preparation was done as follows: 1 μL sample solution wasmixed with 10 μL matrix solution (alpha-cyano-4-hydroxy-cinnamic aciddissolved in a 5:4:1 (v/v/v) mixture of acetonitrile:water:3% (v/v) TFA)and 1 μL was deposited on the sample plate and allowed to dry.Calibration was performed using external standards and the accuracy ofthe mass determinations is within 0.1%. The mass found for the isolatedCART(55-102) was 5257.1 as compared to a calculated mass of 5255.5.

Example 17

[0223] Test for Appetite Suppression in Mice by Recombinant RatCART(55-102, Long), SEQ ID No. 4

[0224] Mice were tested for appetite suppression as described in Example9 after treatment with 0.1-1.0 micrograms of a test substance consistingof recombinant CART(55-102) dissolved in phosphate buffered saline.Intra-cerebroventricular injections of the test substance produced astatistically significant suppression of milk consumption. Dose(micrograms) 0.1 0.2 0.5 1.0 % Feeding Suppression 38% 61% 98% 99%

[0225] The human CART(55-102) peptide corresponding to SEQ ID No. 5differs from the rat form by having a valine residue in stead of aisoleucine residue in position 1. It may be prepared using the samemethod as described for the rat form starting from a human tissue orsimply by substituting valine for isoleucine in position 1 of the ratform.

Example 18

[0226] Test for Appetite Suppression in Mice by Fragmented RecombinantRat CART(55-102, Long), SEQ ID No. 4

[0227] Mice were tested for appetite suppression as described in Example9 after treatment with 0.1-2.0 μg of a test substance consisting offragmented recombinant rat CART(55-102) (fragmented by trypsin andendopeptidase Asp-N) dissolved in phosphate buffered saline.Intra-cerebroventricular injections of the test substance did notproduce statistically significant suppression of milk consumption at anydose tested (see table). Dose (micrograms) 0.1 0.2 0.5 1 2 % FeedingSuppression 1% 0% 3% 0% 0%

Example 19

[0228] Test for Appetite Suppression in Mice by Recombinant RatCART(55-102, Long), SEQ ID No. 4 With a Disrupted Secondary Structure

[0229] Mice were tested for appetite suppression as described in Example9 after treatment with 0.1-2.0 μg of a test substance consisting ofrecombinant rat CART(55-102) with a disrupted secondary structure(reduced and pyridylated) dissolved in phosphate buffered saline.Intra-cerebroventricular injections of the test substance did notproduce statistically significant suppression of milk consumption at anydose tested (see table). Dose (micrograms) 0.1 0.2 0.5 1 2 % FeedingSuppression 9% 12% 0% 26% 18%

Example 20

[0230] Test Method for Measuring Appetite Suppression AfterIntra-Cerebral Injection of a Test Substance in Rats

[0231] Male Wistar rats were implanted with a guide cannula in thelateral cerebral ventricle and allowed to recover for 4-8 days beforescreening for functional cannulae. This was accomplished by injection of5 μg of porcine neuropeptide Y (NPY) , which stimulates feeding withintra-cerebral administration. Animals not responding to NPY werediscarded, and the remaining rats were sorted into response-equivalentgroups of 5-6 rats each. To test the appetite suppressing effects ofcompounds, the rats were first food deprived for 24 hours and thenreceived a 5 μl intra-cerebroventricular injection of a test substancedissolved in phosphate buffered saline (PBS). A control group injectedwith 5 μl of PBS provided reference data for each experiment.Consumption of a special test food (a mash made from a 2:1 mixture ofwater and dry standard chow) was measured for one hour following theinjection. The degree of appetite suppression-produced by a given testsubstance was determined by statistical comparison of the amount of foodconsumed by control rats (vehicle treated) with that of rats treatedwith a test substance. The degree of feeding suppression in a group ofrats was expressed as percent reduction of consumption relative to themean amount consumed by the control group.

Example 21

[0232] Test for Appetite Suppression in Rats by Recombinant RatCART(55-102, Long), SEQ ID No. 4

[0233] Rats were tested for appetite suppression as described in Example20 after treatment with 1 μg of a test substance consisting ofrecombinant rat CART(55-102) dissolved in phosphate buffered saline.Intra-cerebroventricular injection of 1 μg of the test substanceproduced a statistically significant 52% suppression of foodconsumption.

Example 22

[0234] Test for Appetite Suppression in Rats by Recombinant RatCART(55-102, Long), SEQ ID No. 4

[0235] Rats were tested for appetite suppression as described in Example20 after treatment with 0.1-3.0 μg of a test substance consisting ofrecombinant rat CART(55-102) dissolved in phosphate buffered saline.Intra-cerebroventricular injection of 0.3 μg and 3.0 μg of the testsubstance produced statistically significant suppression of foodconsumption (see table). Dose (micrograms) 0.1 0.3 1 3 % FeedingSuppression 17% 45% 19% 63%

[0236]

1 11 102 amino acids amino acid linear peptide 1 Gln Glu Asp Ala Glu LeuGln Pro Arg Ala Leu Asp Ile Tyr Ser Ala 1 5 10 15 Val Asp Asp Ala SerHis Glu Lys Glu Leu Pro Arg Arg Gln Leu Arg 20 25 30 Ala Pro Gly Ala ValLeu Gln Ile Glu Ala Leu Gln Glu Val Leu Lys 35 40 45 Lys Leu Lys Ser LysArg Ile Pro Ile Tyr Glu Lys Lys Tyr Gly Gln 50 55 60 Val Pro Met Cys AspAla Gly Glu Gln Cys Ala Val Arg Lys Gly Ala 65 70 75 80 Arg Ile Gly LysLeu Cys Asp Cys Pro Arg Gly Thr Ser Cys Asn Ser 85 90 95 Phe Leu Leu LysCys Leu 100 89 amino acids amino acid linear protein 2 Gln Glu Asp AlaGlu Leu Gln Pro Arg Ala Leu Asp Ile Tyr Ser Ala 1 5 10 15 Val Asp AspAla Ser His Glu Lys Glu Leu Ile Glu Ala Leu Gln Glu 20 25 30 Val Leu LysLys Leu Lys Ser Lys Arg Ile Pro Ile Tyr Glu Lys Lys 35 40 45 Tyr Gly GlnVal Pro Met Cys Asp Ala Gly Glu Gln Cys Ala Val Arg 50 55 60 Lys Gly AlaArg Ile Gly Lys Leu Cys Asp Cys Pro Arg Gly Thr Ser 65 70 75 80 Cys AsnSer Phe Leu Leu Lys Cys Leu 85 89 amino acids amino acid linear protein3 Gln Glu Asp Ala Glu Leu Gln Pro Arg Ala Leu Asp Ile Tyr Ser Ala 1 5 1015 Val Asp Asp Ala Ser His Glu Lys Glu Leu Ile Glu Ala Leu Gln Glu 20 2530 Val Leu Lys Lys Leu Lys Ser Lys Arg Val Pro Ile Tyr Glu Lys Lys 35 4045 Tyr Gly Gln Val Pro Met Cys Asp Ala Gly Glu Gln Cys Ala Val Arg 50 5560 Lys Gly Ala Arg Ile Gly Lys Leu Cys Asp Cys Pro Arg Gly Thr Ser 65 7075 80 Cys Asn Ser Phe Leu Leu Lys Cys Leu 85 48 amino acids amino acidlinear protein 4 Ile Pro Ile Tyr Glu Lys Lys Tyr Gly Gln Val Pro Met CysAsp Ala 1 5 10 15 Gly Glu Gln Cys Ala Val Arg Lys Gly Ala Arg Ile GlyLys Leu Cys 20 25 30 Asp Cys Pro Arg Gly Thr Ser Cys Asn Ser Phe Leu LeuLys Cys Leu 35 40 45 48 amino acids amino acid linear protein 5 Val ProIle Tyr Glu Lys Lys Tyr Gly Gln Val Pro Met Cys Asp Ala 1 5 10 15 GlyGlu Gln Cys Ala Val Arg Lys Gly Ala Arg Ile Gly Lys Leu Cys 20 25 30 AspCys Pro Arg Gly Thr Ser Cys Asn Ser Phe Leu Leu Lys Cys Leu 35 40 45 49amino acids amino acid linear protein 6 Arg Ile Pro Ile Tyr Glu Lys LysTyr Gly Gln Val Pro Met Cys Asp 1 5 10 15 Ala Gly Glu Gln Cys Ala ValArg Lys Gly Ala Arg Ile Gly Lys Leu 20 25 30 Cys Asp Cys Pro Arg Gly ThrSer Cys Asn Ser Phe Leu Leu Lys Cys 35 40 45 Leu 42 amino acids aminoacid linear peptide 7 Lys Tyr Gly Gln Val Pro Met Cys Asp Ala Gly GluGln Cys Ala Val 1 5 10 15 Arg Lys Gly Ala Arg Ile Gly Lys Leu Cys AspCys Pro Arg Gly Thr 20 25 30 Ser Cys Asn Ser Phe Leu Leu Lys Cys Leu 3540 41 amino acids amino acid linear peptide 8 Tyr Gly Gln Val Pro MetCys Asp Ala Gly Glu Gln Cys Ala Val Arg 1 5 10 15 Lys Gly Ala Arg IleGly Lys Leu Cys Asp Cys Pro Arg Gly Thr Ser 20 25 30 Cys Asn Ser Phe LeuLeu Lys Cys Leu 35 40 35 amino acids amino acid linear protein 9 Cys AspAla Gly Glu Gln Cys Ala Val Arg Lys Gly Ala Arg Ile Gly 1 5 10 15 LysLeu Cys Asp Cys Pro Arg Gly Thr Ser Cys Asn Ser Phe Leu Leu 20 25 30 LysCys Leu 35 80 amino acids amino acid linear protein 10 Ala Leu Asp IleTyr Ser Ala Val Asp Asp Ala Ser His Glu Lys Glu 1 5 10 15 Leu Ile GluAla Leu Gln Glu Val Leu Lys Lys Leu Lys Ser Lys Arg 20 25 30 Ile Pro IleTyr Glu Lys Lys Tyr Gly Gln Val Pro Met Cys Asp Ala 35 40 45 Gly Glu GlnCys Ala Val Arg Lys Gly Ala Arg Ile Gly Lys Leu Cys 50 55 60 Asp Cys ProArg Gly Thr Ser Cys Asn Ser Phe Leu Leu Lys Cys Leu 65 70 75 80 80 aminoacids amino acid linear protein 11 Ala Leu Asp Ile Tyr Ser Ala Val AspAsp Ala Ser His Glu Lys Glu 1 5 10 15 Leu Ile Glu Ala Leu Gln Glu ValLeu Lys Lys Leu Lys Ser Lys Arg 20 25 30 Val Pro Ile Tyr Glu Lys Lys TyrGly Gln Val Pro Met Cys Asp Ala 35 40 45 Gly Glu Gln Cys Ala Val Arg LysGly Ala Arg Ile Gly Lys Leu Cys 50 55 60 Asp Cys Pro Arg Gly Thr Ser CysAsn Ser Phe Leu Leu Lys Cys Leu 65 70 75 80

1. An isolated polypeptide comprising the sequence SEQ ID No. 1:Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Pro-Arg-Arg-Gln-Leu-Arg-Ala-Pro-Gly-Ala-Val-Leu-Gln-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser- Phe-Leu-Leu-Lys-Cys-Leu

in which the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe N-terminal end.
 2. An isolated polypeptide comprising the sequenceSEQ ID No. 2: Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-IIe-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe- Leu-Leu-Lys-Cys-Leu

in which the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe n-terminal end:
 3. An isolated polypeptide comprising the sequenceSEQ ID No. 3: Gln-Glu-Asp-Ala-Glu-Leu-Gln-Pro-Arg-Ala-Leu-Asp-Ile-Tyr-Ser-Ala-Val-Asp-Asp-Ala-Ser-His-Glu-Lys-Glu-Leu-Ile-Glu-Ala-Leu-Gln-Glu-Val-Leu-Lys-Lys-Leu-Lys-Ser-Lys-Arg-Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Cly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe- Leu-Leu-Lys-Cys-Leu

in which the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe N-terminal end.
 4. An isolated polypeptide comprising the sequenceSEQ ID No. 4: Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu

in which the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe N-terminal end.
 5. An isolated polypeptide comprising the sequenceSEQ ID No. 5: Val-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu


6. An isolated polypeptide comprising the sequence SEQ ID No. 6:Arg-Ile-Pro-Ile-Tyr-Glu-Lys-Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys- Leu


7. An isolated polypeptide comprising the sequence SEQ ID No. 7:Lys-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser- Phe-Leu-Leu-Lys-Cys-Leu


8. An isolated polypeptide comprising the sequence SEQ ID No. 8:Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe- Leu-Leu-Lys-Cys-Leu


9. An isolated polypeptide comprising the sequence SEQ ID No. 9:Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu


10. An isolated polypeptide according to any one of the claims 5 to 9 inwhich the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe N-terminal end.
 11. A nucleic acid construct comprising a nucleotidesequence encoding a CART polypeptide or a fragment or variant thereofwith appetite regulating activity/function.
 12. A nucleic acid constructaccording to claim 11 comprising a nucleotide sequence encoding apolypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to9.
 13. A nucleic acid construct according to claim 12 comprising anucleotide sequence encoding a polypeptide with a sequence selected fromthe sequences SEQ ID Nos. 1 to 9 in which the cysteine residues arelinked by disulphide bonds in the configuration I-III, II-V and IV-VIwhen the cysteines are numbered from the N-terminal end.
 14. Arecombinant vector comprising the nucleic acid construct according toany one of the claims 11 to
 13. 15. A recombinant host cell comprisingthe nucleic acid construct according to any one of the claims 11 to 13or the vector according to claim
 14. 16. The cell according to claim 15,which is of mammalian, insect, plant, microbial, bacterial, or fungalorigin.
 17. A transgenic animal containing and expressing the nucleicacid construct according to any one of the claims 11 to
 13. 18. Atransgenic plant containing and expressing the nucleic acid constructaccording to any one of the claims 11 to
 13. 19. A method of producing aCART polypeptide or a fragment or variant thereof with appetiteregulating activity/function, which method comprises cultivating a cellaccording to claim 15 or 16 in a suitable culture medium underconditions permitting expression of the nucleic acid construct andrecovering the resulting polypeptide from the culture medium/cell.
 20. Amethod of producing a CART polypeptide or a fragment or variant thereofwith appetite regulating activity/function, which method comprisesrecovering the polypeptide produced by the transgenic animal accordingto claim
 17. 21. A method of producing a CART polypeptide or a fragmentor variant thereof with appetite regulating activity/function, whichmethod comprises growing a cell of a transgenic plant according to claim18, and recovering the polypeptide from the resulting plant.
 22. Anantibody capable of specifically binding to a CART polypeptide or afragment or variant thereof with appetite regulating activity/function.23. An antibody capable of specifically binding to a polypeptide with asequence selected from the sequences SEQ ID Nos. 1 to
 9. 24. An antibodycapable of specifically binding to a polypeptide with a sequenceselected from the sequences SEQ ID Nos. 1 to 9 in which the cysteineresidues are linked by disulphide bonds in the configuration I-III, II-Vand IV-VI when the cysteines are numbered from the N-terminal end. 25.An antibody according to any one of the claims 22 to 24 which is amonoclonal antibody.
 26. A hybridoma which produces a monoclonalantibody according to claim
 25. 27. An appetite regulating compositioncomprising a CART polypeptide or a fragment or variant thereof and apharmaceutical acceptable carrier.
 28. An appetite regulatingcomposition according to claim 27 comprising a polypeptide with asequence selected from the sequences SEQ ID Nos. 1 to 9 and apharmaceutical acceptable carrier.
 29. An appetite regulatingcomposition according to claim 28 comprising a polypeptide with asequence selected from the sequences SEQ ID Nos. 1 to 9 in which thecysteine residues are linked by disulphide bonds in the configurationI-III, II-V and IV-VI when the cysteines are numbered from theN-terminal end and a pharmaceutical acceptable carrier.
 30. Use of aCART polypeptide or a fragment or variant thereof for the preparation ofa medicament for the regulation of appetite.
 31. Use of a polypeptidewith a sequence selected from the sequences SEQ ID Nos. 1 to 9 for thepreparation of a medicament for the regulation of appetite.
 32. Use of apolypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to9 in which the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe N-terminal end for the preparation of a medicament for theregulation of appetite.
 33. Use of a CART polypeptide or a fragment orvariant thereof for the preparation of a medicament for the treatment ofobesity.
 34. Use of a polypeptide with a sequence selected from thesequences SEQ ID Nos. 1 to 9 for the preparation of a medicament for thetreatment of obesity.
 35. Use of a polypeptide with a sequence selectedfrom the sequences SEQ ID Nos. 1 to 9 in which the cysteine residues arelinked by disulphide bonds in the configuration I-III, II-V and IV-VIwhen the cysteines are numbered from the N-terminal end for thepreparation of a medicament for the treatment of obesity.
 36. A methodfor the regulation of appetite comprising administering to an subject inneed thereof an effective amount of an isolated CART polypeptide or afragment or variant thereof.
 37. A method for the regulation of appetitecomprising administering to an subject in need thereof an effectiveamount of a polypeptide with a sequence selected from the sequences SEQID Nos. 1 to
 9. 38. A method for the regulation of appetite comprisingadministering to an subject in need thereof an effective amount of apolypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to9 in which the cysteine residues are linked by disulphide bonds in theconfiguration I-III, II-V and IV-VI when the cysteines are numbered fromthe N-terminal end.
 39. Any novel feature or combination of featuresdescribed herein.